Compositions and methods for differentiation of human pluripotent stem cells into desired cell types

ABSTRACT

In related-art methods of differentiating pluripotent stem cells into a desired cell type, there has not been established a differentiation induction method using human ES/iPS cells and being stable and highly efficient. The use of complicated culture steps is a large problem. In addition, there are also large problems in, for example, that the speed of cell differentiation is low, and hence long-period culture is required, and that the differentiation efficiency is low, and hence it is difficult to obtain a sufficient number of required cells. A method of inducing differentiation into a desired cell type, which induces differentiation within a short period of time and with high efficiency by the use of a Sendai virus vector capable of expressing a transcription factor, and as required, the use of a pluripotent stem cell in which an expression amount of a POU5F1 protein has been substantially removed or reduced, is provided.

The present application claims priority from U.S. Provisional PatentApplication No. 62/465,188 and U.S. Provisional Patent Application No.62/523,324, which are incorporated herein by reference.

BACKGROUND OF THE INVENTION 1. Field of the Invention

The present invention relates to a method of efficiently differentiatinga pluripotent stem cell into a desired cell type.

2. Description of the Related Art

Human pluripotent stem cells (hPSCs), such as human embryonic stem cells(hESCs) and human induced pluripotent stem cells (hiPSCs), havepotentials to become all cell types in a human body in vitro. For thelast 20 years, use of neurons, muscles, and other cell typesdifferentiated from hPSCs for cell transplantation therapy and drugscreening has been widely investigated, and practical applicationthereof has been under consideration. However, the practical applicationis limited, and one of the major obstacles is difficulty ofdifferentiating hPSCs into desired cell types, such as neurons andmuscles.

The most generally used method is step-by-step differentiation based onsuccessive changes in cell culture environment (Hu et al., 2010, PNAS107, 4335-4340). Those differentiation steps have been well establishedand widely used, but have fundamental limitations in both speed andscale. Further, the process is relatively slow, and takes several weeksuntil formation of functional neurons as the desired cell type.

As another method, PSCs can be rapidly and efficiently differentiated byinducing or overexpressing transcription factors (TFs) with plasmids,viruses, and other vectors (Busskamp et al., 2014, Molecular SystemsBiology 10, 760; Hester et al., Molecular Therapy, 19, 1905-1912; Zhanget al., 2013, Neuron, 78, 785-798).

The inventors of the present invention have reported rapid and efficientdifferentiation into skeletal muscles (Akiyama et al., 2016, Development143, 3674), motor neurons (Goparaju et al., 2017, Scientific Reports 7,42367), and lacrimal gland epithelium-like cells (Hirayama et al., npjAging and Mechanisms of Disease 2017, 1) by transfection of a cocktailof synthetic mRNAs encoding TFs into hPSCs. The differentiation methodbased on synthetic mRNA has many desirable features. mRNA is notincorporated into a cellular genome, and hence achieves safe andfootprint-free transfer of a gene product. However, this method requiresa plurality of times of transfection of synthetic mRNAs into cells,typically one or two times of transfection a day for several consecutivedays. Thus, this method puts a huge burden on an experimenter, andrequires expert skills.

In related-art methods of differentiating pluripotent stem cells into adesired cell type, there has not been established a differentiationinduction method using human ES/iPS cells and being stable and highlyefficient. Many attempts have been made, which includes a step-by-stepdifferentiation induction method based on the control of cultureconditions or the addition of, for example, various cell growthfactors/differentiation factors to a culture solution, but the use ofcomplicated culture steps is a big problem. In addition, there are alsobig problems in, for example, that the speed of cell differentiation islow, and hence long-period culture is required, and that thedifferentiation efficiency is low, and hence it is difficult to obtain asufficient number of required cells.

SUMMARY OF THE INVENTION

The inventors of the present invention have developed a method ofinducing differentiation into a desired cell type within a short periodof time and with high efficiency by the use of a Sendai virus vectorcapable of expressing a transcription factor, and as required, the useof a pluripotent stem cell in which an expression amount of a POU5F1protein has been substantially removed or reduced.

Thus, the present invention has been completed.

That is, the present invention includes the following.

1. A method of differentiating a pluripotent stem cell into a desiredcell type, including:

1) adding a Sendai virus vector containing a transcription factorrequired for induction of differentiation into the desired cell type toa pluripotent stem cell in a cell culture medium; and

2) culturing the pluripotent stem cell to differentiate the pluripotentstem cell into the desired cell type.

2. A method according to the above-mentioned item 1, further includingadding a gene for a compound having an action of substantially removingor reducing an expression amount of a POU5F1 protein to the pluripotentstem cell.

3. A method according to the above-mentioned item 2, wherein the genefor the compound having an action of substantially removing or reducingan expression amount of a POU5F1 protein includes siRNA against POU5F1.

4. A method according to the above-mentioned item 1, in which thepluripotent stem cell includes a pluripotent stem cell in which anexpression amount of a POU5F1 protein has been substantially removed orreduced.

5. A method according to any one of the above-mentioned items 1 to 4,wherein the adding a Sendai virus vector containing a transcriptionfactor required for induction of differentiation into the desired celltype is performed once.

6. A method according to any one of the above-mentioned items 1 to 5,wherein the Sendai virus vector is temperature-sensitive.

7. A method according to any one of the above-mentioned items 1 to 6,wherein the desired cell type includes a skeletal muscle cell, andwherein the transcription factor includes MYOD1.

8. A method according to the above-mentioned item 7, whereindifferentiation efficiency of the skeletal muscle cell is 75% or more.

9. A method according to the above-mentioned item 7 or 8, whereindifferentiation efficiency of the skeletal muscle cell is 75% or more,and wherein one kind of cell culture medium is used.

10. A method according to any one of the above-mentioned items 1 to 6,wherein the desired cell type includes a motor neuron, and wherein thetranscription factor includes NEUROG3.

11. A method according to the above-mentioned item 10, whereindifferentiation efficiency of the motor neuron is about 90%.

12. A method according to the above-mentioned item 10 or 11, whereindifferentiation efficiency of the motor neuron is about 90%, and whereinone kind of cell culture medium is used.

13. A method according to any one of the above-mentioned items 1 to 6,wherein the desired cell type includes a liver cell, and wherein thetranscription factor includes FOXA1 and HNF1A.

14. A method according to the above-mentioned item 13, wherein two kindsof cell culture media are used.

15. A method according to any one of the above-mentioned items 1 to 6,wherein the desired cell type includes a hematopoietic cell, and whereinthe transcription factor includes SPI1.

16. A method according to any one of the above-mentioned items 1 to 6,wherein the desired cell type includes a dopaminergic neuron, andwherein the transcription factor includes FOXA1.

The method of efficiently differentiating a pluripotent stem cell into adesired cell type of the present invention has at least any one or moreof the following effects.

(1) The period of time required for cell differentiation starting withthe pluripotent stem cell is shortened and the differentiation inductionefficiency is improved.

(2) Differentiation can be performed by adding the transcription factorrequired for induction of differentiation into the desired cell typeonly once.

(3) The number of kinds of transcription factors required for inductionof differentiation into the desired cell type can be decreased ascompared to related-art methods.

(4) When the method is combined with a method of reducing theundifferentiation maintenance of a pluripotent stem cell and/or a methodof reducing the differentiation resistance thereof, the period of timerequired for cell differentiation starting with the pluripotent stemcell is shortened and the differentiation induction efficiency isimproved.

BRIEF DESCRIPTION OF THE DRAWINGS

The patent or application file contains at least one drawing executed incolor. Copies of this patent or patent application publication withcolor drawing(s) will be provided by the Office upon request and paymentof the necessary fee.

FIG. 1A, FIG. 1B, and FIG. 1C are a schematic and images forillustrating and showing differentiation into skeletal muscles. FIG. 1Ais a schematic of typical experimental steps. On Day 1, human iPS cellswere plated on a cell culture dish. Immediately after the plating, aSendai virus vector encoding a human MYOD1 gene was added to the cellculture medium. The cells were cultured in a CO₂ incubator at 33° C. OnDay 2, siPOU5F1 was added to the cell culture medium. On Day 4, thetemperature of the CO₂ incubator was changed to 37° C. On Day 6, inorder to evaluate the efficiency of cell differentiation, the cells werefixed, and used for immunostaining. FIG. 1B is a microscopic image (10×objective lens) of cells immunostained with anti-myosin heavy chain (redsignal), which is specific for mature skeletal muscles. The cells werefurther stained with DAPI (green signal) for visualizing the nuclei ofall cells. FIG. 1C is a microscopic image (20× objective lens) of cellsimmunostained with anti-myosin heavy chain (red signal), which isspecific for mature skeletal muscles. The cells were further stainedwith DAPI (green signal) for visualizing the nuclei of all cells.

FIG. 2A, FIG. 2B, FIG. 2C, FIG. 2D, and FIG. 2E are a schematic andimages for illustrating and showing differentiation into cholinergicand/or motor neurons. FIG. 2A is a schematic of typical experimentalsteps. On Day 1, human iPS cells were plated on a cell culture dish.Immediately after the plating, a Sendai virus vector encoding a humanNGN3 gene was added to the cell culture medium. The cells were culturedin a CO₂ incubator at 33° C. On Day 3, the temperature of the CO₂incubator was changed to 37° C. On Day 4, the cells were re-plated on aglass coverslip. On Day 6, in order to evaluate the efficiency of celldifferentiation, the cells were fixed, and used for immunostaining. FIG.2B is a microscopic image (20× objective lens) of cells stained withDAPI (blue signal) for visualizing the nuclei of all cells. FIG. 2C is amicroscopic image (20× objective lens) of cells immunostained withanti-β3-tubulin (TUBB3) (red signal), which is specific for matureneurons. FIG. 2D is a microscopic image (20× objective lens) of cellsimmunostained with anti-choline acetyltransferase (ChAT) antibody (greensignal), which is specific for mature motor neurons. FIG. 2E is asynthetic image of FIG. 2C and FIG. 2D.

FIG. 3A, FIG. 3B, FIG. 3C, FIG. 3D, and FIG. 3E are a schematic andimages for illustrating and showing differentiation into liver cells.FIG. 3A is a schematic of typical experimental steps. On Day 1, humaniPS cells were plated on a cell culture dish. Immediately after theplating, a mixture of equal amounts of a Sendai virus vector encoding ahuman FOXA1 gene and a Sendai virus vector encoding a human HNF1A genewas added to the cell culture medium. The cell culture medium is aROCK-inhibitor (Y27632)-containing StemFit (registered trademark inJapan) Basic02 (Ajinomoto). The cells were cultured in a CO₂ incubatorat 33° C. On Day 2, siPOU5F1 was added to the cell culture medium. Thecell culture medium was replaced with a differentiation medium. On Day5, the temperature of the CO₂ incubator was changed to 37° C. On Day 7,the cell culture medium was replaced with a maturation medium. On Day 8,in order to evaluate the efficiency of cell differentiation, the cellswere fixed, and used for immunostaining. FIG. 3B is a microscopic image(10× objective lens) of cells immunostained with anti-α-fetoprotein(AFP) antibody (red signal), which is a fetal form of albumin and isspecific for embryonic liver cells. FIG. 3C is a microscopic image (10×objective lens) of cells immunostained with anti-albumin (ALB) antibody(green signal), which is specific for liver cells. FIG. 3D is amicroscopic image (20× objective lens) of cells immunostained withanti-α-fetoprotein (AFP) antibody (red signal), which is a fetal form ofalbumin and is specific for embryonic liver cells. FIG. 3E is amicroscopic image (20× objective lens) of cells immunostained withanti-albumin (ALB) antibody (green signal), which is specific for livercells.

DESCRIPTION OF THE EMBODIMENTS

(The Present Invention)

A method of efficiently differentiating a pluripotent stem cell into adesired cell type of the present invention (hereinafter sometimesreferred to as “method of the present invention”) includes part or allof the following steps, though the method is not particularly limited aslong as the method uses a Sendai virus vector capable of expressing atranscription factor required for induction of differentiation into thedesired cell type:

1) a step of adding a Sendai virus vector containing a transcriptionfactor required for induction of differentiation into the desired celltype to a pluripotent stem cell in a cell culture medium; and

2) a step of culturing the pluripotent stem cell to differentiate thepluripotent stem cell into the desired cell type.

The adding of various compounds, transcription factors, vectors and thelike to the pluripotent stem cell encompasses addition to a medium inwhich the pluripotent stem cell is present.

(Pluripotent Stem Cell)

The pluripotent stem cell to be used in the method of the presentinvention is not particularly limited, but is preferably derived from amammal, particularly preferably derived from a human. The pluripotentstem cell is, for example, a human ES cell, a human iPS cell, or anycombination thereof, is not particularly limited, and encompasses tissuestem cells derived from tissues and organs, dermal fibroblasts, and allkinds of cells derived from tissues and organs.

(Sendai Virus Vector)

Sendai virus is a kind of virus of the genus Respirovirus of the familyParamyxoviridae, and has single-stranded RNA as its genes.

The Sendai virus vector to be used in the method of the presentinvention may be a natural strain, a wild-type strain, a mutant strain,or a commercially available product (e.g., from ID Pharma).

An example thereof may be an F gene-deleted Sendai virus vector havingG69E, T116A, and A183S mutations in M protein, A262T, G264R, and K461Gmutations in HN protein, D433A, R434A, K437A, and L511F mutations in Pprotein, and L1361C, L1558I, N1197S, and K1795E mutations in L protein.

In addition, the Sendai virus vector to be used in the method of thepresent invention is preferably a temperature-sensitive strain. The term“temperature-sensitive” refers to a significant lowering of activity ata general cell culture temperature (e.g., from 37° C. to 38° C.) ascompared to low temperature (e.g., from 30° C. to 36° C.). For example,mutations such as TS 7 (Y942H/L1361C/L1558I mutations in the L protein),TS 12 (D433A/R434A/K437A mutations in the P protein), TS 13(D433A/R434A/K437A mutations in the P protein and an L1558I mutation inthe L protein), TS 14 (D433A/R434A/K437A mutations in the P protein andan L1361C mutation in the L protein), and TS 15 (D433A/R434A/K437Amutations in the P protein and L1361C/L1558I mutations in the L protein)of the Sendai virus are temperature-sensitive mutations, and may besuitably utilized in the present invention.

For those Sendai virus vectors, reference may be made to Japanese PatentRe-publication No. 2015/046229.

(Transcription Factor Required for Induction of Differentiation intoDesired Cell Type)

The form of the “transcription factor required for induction ofdifferentiation into the desired cell type” to be used in the method ofthe present invention is not particularly limited as long as thetranscription factor can be carried on the Sendai virus vector, butexamples thereof may include, but not particularly limited to, nucleicacids, such as RNA and DNA, and synthetic nucleic acids.

In addition, in the method of the present invention, examples of thedesired cell type may include skeletal muscles (skeletal muscle cells),nerve cells (motor neurons and dopaminergic neurons), the liver (livercells), chondrocytes, bone cells, and hematopoietic cells.

As described in Examples below, in the method of the present invention,differentiation can be performed by adding the transcription factorrequired for induction of differentiation into the desired cell typeonly once.

Further, as described in Examples below, in the method of the presentinvention, the number of kinds of required transcription factors hasbeen successfully reduced as compared to related-art methods by carryingthe transcription factor required for induction of differentiation intothe desired cell type on the Sendai virus vector.

In addition, as described in Examples below, in the method of thepresent invention, the differentiation efficiency has been successfullyimproved as compared to related-art methods by carrying thetranscription factor required for induction of differentiation into thedesired cell type on the Sendai virus vector.

Besides, as described in Examples below, in the method of the presentinvention, the number of kinds of required transcription factors hasbeen successfully reduced and the differentiation efficiency has beensuccessfully improved as compared to related-art methods by: carryingthe transcription factor required for induction of differentiation intothe desired cell type on the Sendai virus vector; and adding a gene fora compound having an action of substantially removing or reducing anexpression amount of a POU5F1 protein to the pluripotent stem cell.

(Method of Efficiently Inducing Differentiation of Pluripotent Stem Cellinto Desired Cell Type of the Present Invention)

In the method of the present invention, a method of reducing theundifferentiation maintenance of a pluripotent stem cell, and asrequired, a method capable of reducing the differentiation resistance ofa pluripotent stem cell to the desired cell type may be preferablyintroduced. Examples thereof may include the following.

(Pluripotent Stem Cell Whose Undifferentiation Maintenance has beenReduced)

In pluripotent stem cells, the expression of a transcription factorPOU5F1 (SEQ ID NOS: and 2: POU domain, class 5, transcription factor 1isoform 1: ncbi.nlm.nih.gov/protein/NP_002692, other names: OCT3, OCT4,OTF3, OTF4, OTF-3, Oct-3, Oct-4, MGC22487) is essential to theundifferentiation maintenance of the pluripotent stem cells. POU5F1 isspecifically expressed in pluripotent cells, such as reproductive cellsand a preimplantation early embryo. That is, the “reducing theundifferentiation maintenance of a pluripotent stem cell” in the presentinvention means substantially removing or reducing an expression amountof a POU5F1 protein in the pluripotent stem cell. The substantiallyremoving or reducing an expression amount of a POU5F1 proteinencompasses inhibiting the process of any one of the transcription andtranslation stages of POU5F1 and/or inhibiting the activity of thetranslated POU5F1 protein, and is not particularly limited.

Besides, a state in which the expression amount of the POU5F1 protein inthe pluripotent stem cell has been substantially removed or reduced maybe confirmed by a comparison to the degree of the expression amount ofthe POU5F1 protein (or expression amount of the POU5F1 gene) in apluripotent stem cell that has not been subjected to the removing or thereducing. For example, the state (degree) in which the expression amountof the POU5F1 protein in the pluripotent stem cell has beensubstantially removed or reduced is from 95 to 1, from 90 to 2, from 85to 3, from 80 to 4, from 75 to 5, from 70 to 6, from 65 to 7, from 60 to8, from 50 to 10, from 40 to 15, from 30 to 20, or about 25 whencompared to the expression amount of the POU5F1 protein in thepluripotent stem cell that has not been removed or reduced, which isdefined as 100. The degree of the expression amount of the POU5F1protein in the pluripotent stem cell may be easily measured by using acommercially available anti-POU5F1 antibody, and the gene expressionamount of POU5F1 may be measured by a method known per se (see WO2017/131238 A1).

(Reducing Differentiation Resistance of Pluripotent Stem Cell to DesiredCell Type)

In pluripotent stem cells, a special chromatin structure called a“bivalent domain” is formed in each promoter region of a group of genesinvolved in differentiation, and under a stemness-maintaining state, thegroup of genes involved in development/differentiation are in a standbystate so as not to be easily expressed. The inventors of the presentinvention have confirmed that “when a methyl group modification of ahistone called H3K27me3 is removed or reduced in the “bivalent domain”,the expression of differentiation genes required for induction ofdifferentiation into the desired cell type is rapidly and efficientlyfacilitated” (see WO 2017/073763 A1).

That is, the “reducing differentiation resistance of a pluripotent stemcell to a desired cell type” of the present invention means that theH3K27me3 modification of the pluripotent stem cell is substantiallyremoved or reduced.

In addition, a state in which the H3K27me3 modification of thepluripotent stem cell has been substantially removed or reduced may beconfirmed by a comparison to the degree of the H3K27me3 modification ofa pluripotent stem cell that has not been subjected to the removing orthe reducing. For example, the state (degree) in which the H3K27me3modification of the pluripotent stem cell has been substantially removedor reduced is from 95 to 1, from 90 to 2, from 85 to 3, from 80 to 4,from 75 to 5, from 70 to 6, from 65 to 7, from 60 to 8, from 50 to 10,from 40 to 20, or about 30 when compared to the degree of the H3K27me3modification of the pluripotent stem cell that has not been removed orreduced, which is defined as 100. The degree of the H3K27me3modification of the pluripotent stem cell may be easily measured byusing a commercially available anti-Histone H3K27me3 antibody, and thegene expression amount of H3K27me3 may be measured by a method known perse.

(Use of Modified Synthetic mRNA for Target Gene)

The method of the present invention preferably includes adding(introducing), to the pluripotent stem cell, a gene for a compoundhaving an action of substantially removing or reducing an expressionamount of a POU5F1 protein (a gene expressing small interfering RNA(siRNA) against POU5F1, a gene expressing shRNA against POU5F1, a geneexpressing an antisense strand of POU5F1, or an antibody gene).

Similarly, the method of the present invention preferably includesadding (introducing), to the pluripotent stem cell, a gene for acompound having an action of substantially removing or reducing H3K27me3modification.

The term “gene” as used herein encompasses not only double-strandednucleic acids, but also their respective constituent single strands,such as plus strands (or sense strands) or complementary strands (orantisense strands), linear nucleic acids, and circular nucleic acids,and encompasses DNA, RNA, mRNA, cDNA, and the like, unless otherwisestated.

Besides, the target gene is meant to encompass the gene for the compoundhaving an action of substantially removing or reducing an expressionamount of a POU5F1 protein, and/or the gene for the compound having anaction of substantially removing or reducing H3K27me3 modification, andthe transcription factor required for induction of differentiation intothe desired cell type.

A method known per se may be used without any particular limitation as amethod of adding (introducing) the gene for the compound having anaction of substantially removing or reducing an expression amount of aPOU5F1 protein or the gene for the compound having an action ofsubstantially removing or reducing H3K27me3 modification to thepluripotent stem cell. There is preferably used a method of inducingdifferentiation by efficiently introducing synthetic mRNA into humanpluripotent stem cells through use of a gene expression method involvingusing synthetic mRNA developed by Warren, Rossi, et al. (reference: CellStem Cell 7: 618-630, 2010.), which is a footprint-free forced geneexpression method causing no gene incorporation into a host genome (seeWO 2017/131238 A1).

The timing, at which the gene for the compound having an action ofsubstantially removing or reducing an expression amount of a POU5F1protein (or the gene for the compound having an action of substantiallyremoving or reducing H3K27me3 modification) and the Sendai virus vectorcontaining the transcription factor required for induction ofdifferentiation into the desired cell type are added to the pluripotentstem cell, is not particularly limited. It is preferred that the genefor the compound having an action of substantially removing or reducingan expression amount of a POU5F1 protein (or the gene for the compoundhaving an action of substantially removing or reducing H3K27me3modification) be added to the pluripotent stem cell after the additionof the Sendai virus vector containing the transcription factor requiredfor induction of differentiation into the desired cell type.

Further, the timing and number of times of the addition of each gene(mRNA) are not particularly limited. In the method of the presentinvention, unlike the related art, differentiation can be performed withhigh efficiency by adding the transcription factor required forinduction of differentiation into the desired cell type (Sendai virusvector containing the transcription factor required for induction ofdifferentiation into the desired cell type), and as required, the genefor the compound having an action of substantially removing or reducingan expression amount of a POU5F1 protein once during culture.

(Synthesis of Modified mRNA Encoding Amino Acid Sequence ofTranscription Factor)

Modified mRNA is synthesized with reference to a method described in theliterature “Warren et al., Cell Stem Cell, 2010 Nov. 5; 7 (5): 618-30.”More specifically, mRNA is synthesized by in vitro transcription using amixture of dNTPs {(dNTPs: 3-O-Me-m⁷G(5′)ppp(5′)G ARCA cap analog,5-methylcytidine triphosphate, and pseudouridine triphosphate)} obtainedby modifying template DNA encoding the amino acid sequence of thetranscription factor required for induction of differentiation into thedesired cell type.

(Generation of Sendai Virus Vector Encoding Transcription FactorRequired for Induction of Differentiation into Desired Cell Type)

In the present invention, the Sendai virus vector is used for theintroduction of the transcription factor required for induction ofdifferentiation into the desired cell type. Further, in order to expressa mammalian (in particular, human) transcription factor, a Sendai virusvector capable of expressing a human transcription factor is preferablyused. In particular, a mutant of a Sendai virus vector, such as an Fprotein-deficient mutant, has no infectivity, and hence is easy tohandle (see Inoue et al., J Virol. 77.5: 3238-3246, 2003).

(Method of Inducing Differentiation of Pluripotent Stem Cell intoDesired Cell Type with High Efficiency)

A single transcription factor or a cocktail of two or more transcriptionfactors required for induction of differentiation into the desired celltype is prepared. The form of the transcription factor is a Sendai virusvector having incorporated therein a transcription factor (or aplurality of transcription factors).

(Use of Expression Vector)

In a step of the method of the present invention, there may be used anexpression vector known per se having introduced therein the gene forthe compound having an action of substantially removing or reducing anexpression amount of a POU5F1 protein (or the gene for the compoundhaving an action of substantially removing or reducing H3K27me3modification) and/or the transcription factor required for induction ofdifferentiation into the desired cell type. An example of the expressionvector to be used in the present invention may be a Sendai virus vector.

A method of introducing the expression vector into the pluripotent stemcell is not particularly limited, but examples thereof may include alipofection method, a liposome method, an electroporation method, acalcium phosphate coprecipitation method, a diethylaminoethyl(DEAE)-dextran method, a microinjection method, and a gene gun method. Aparticularly preferred example is a lipofection method.

As another method, the gene for the compound having an action ofsubstantially removing or reducing an expression amount of a POU5F1protein (or the gene for the compound having an action of substantiallyremoving or reducing H3K27me3 modification) may be converted to cationicsiRNA by binding spermine, phosphospermine, or the like thereto. Thecationic siRNA does not require a reagent for transfection.

(Compound Having Action of Substantially Removing or Reducing ExpressionAmount of POU5F1 Protein)

The compound having an action of substantially removing or reducing anexpression amount of a POU5F1 protein of the present invention is notparticularly limited, but is, for example, siRNA against POU5F1, shRNAagainst POU5F1, an antisense strand of POU5F1, an antibody thatspecifically binds to the POU5F1 protein, or an inhibitor.

In addition, not only by using those compounds alone, but by using aplurality of kinds of compounds and/or a low-molecular-weight compoundin combination, it is possible to efficiently “reduce theundifferentiation maintenance of a pluripotent stem cell (substantiallyremove or reduce an expression amount of a POU5F1 protein of apluripotent stem cell).”

(Compound Having Action of Substantially Removing or Reducing H3K27Me3Modification)

The compound having an action of substantially removing or reducingH3K27me3 modification of the present invention is not particularlylimited, but is, for example, a demethylase (in particular, ademethylase having an action of removing a methyl group of H3K27me3), anantibody that specifically binds to H3K27me3, an antibody forPolycomb-group proteins (PcG proteins) having an H3K27me3 modificationaction, siRNA (in particular, cationic siRNA), or an inhibitor. Thecationic siRNA does not require a reagent for transfection.

Examples of the compound may include low-molecular-weight compoundsincluding, but not particularly limited to, histone deacetylase (HDAC)inhibitors, such as valproic acid.

(JMJD3)

JMJD3 is known as a demethylase for H3K27me3 of a histone (mouseNP_001017426, human NP_001073893), and even in its full length(NP_001073893, SEQ ID NO: 3), has an action of substantially removing orreducing the H3K27me3 modification of pluripotent stem cells. Theinventors of the present invention have confirmed that JMJD3c having theJmjC domain {SEQ ID NO: 4, catalytic domain: SEQ ID NO: 5 (amino acids1376-1484)} has a stronger action of substantially removing or reducingH3K27me3 modification as compared to full-length JMJD3 (see WO2017/073763 A1).

A preferred base sequence of JMJD3 is a base sequence set forth in SEQID NO: 6.

(Kind of Transcription Factor Required for Induction of Differentiationinto Desired Cell Type)

The form of the “transcription factor required for induction ofdifferentiation into the desired cell type” to be used in the method ofthe present invention is not particularly limited as long as thetranscription factor can be carried on the Sendai virus vector, butexamples thereof may include, but not particularly limited to, nucleicacids, such as RNA and DNA, and synthetic nucleic acids.

In addition, in the method of the present invention, examples of thedesired cell type may include skeletal muscles (skeletal muscle cells),the liver (liver cells), nerve cells (motor neurons and dopaminergicneurons), chondrocytes, bone cells, and hematopoietic cells.

As described in Examples below, in the method of the present invention,the number of kinds of transcription factors required for induction ofdifferentiation into the desired cell type has been successfully reducedand/or high differentiation efficiency has been successfully achieved bycarrying the transcription factor required for induction ofdifferentiation into the desired cell type on the Sendai virus vector.

{Transcription Factor Required for Induction of Differentiation intoSkeletal Muscle (in Particular, Cells Present in Skeletal Muscle)}

A method of inducing differentiation into a skeletal muscle is asdescribed below.

A single transcription factor, or two or more transcription factorsselected from the group consisting of MYOD1, NRF1, SALL4, ZIC1, KLF9,ZNF281, CTCF, HES1, HOXA2, TBX5, TP73, ERG, MAB21L3, PRDM1, NFIC, CTCFL,FOXP1, HEY1, PITX2, JUNB, KLF4, ESX1, TFAP2C, FOS, TFE3, FOSL1, GRHL2,TBX2, NFIB, and IRF4 are introduced into pluripotent stem cells. Inparticular, MYOD1 (base sequence: SEQ ID NO: 7, amino acid sequence: SEQID NO: 8) is preferably introduced alone into pluripotent stem cells.

(Transcription Factor Required for Induction of Differentiation intoNerve Cells)

A method of inducing differentiation into nerve cells (in particular,motor neurons or dopaminergic neurons) is as described below.

A single transcription factor, or two, three, four, five, or sixtranscription factors selected from NEUROG1, NEUROG2, NEUROG3, NEUROD1,NEUROD2, and FOXA1 are introduced into human pluripotent stem cells.

For example, for the motor neurons (cholinergic and/or motor neurons),NEUROG3 (NGN3) (base sequence: SEQ ID NO: 9, amino acid sequence: SEQ IDNO: 10) is preferably introduced alone into pluripotent stem cells.

For example, for the dopaminergic neurons, FOXA1 (accession number: NM004496) is preferably introduced alone into pluripotent stem cells.

{Transcription Factor Required for Induction of Differentiation intoLiver (in Particular, Cells Present in Liver, i.e., Hepatoblasts)}

A method of inducing differentiation into the liver (in particular, theliver or the fetal liver) is as described below.

For the liver, only one transcription factor, or two or moretranscription factors selected from HNF1A, TCF-1, SALL4, TGIF1, MAB21L3,ZIC1, EGFLAM, PITX2, HNF4A, NRF1, ZNF281, CTCFL, TP73, TFE3, DLX6, andTCF4 are introduced into human pluripotent stem cells.

For the fetal liver, only one transcription factor, or two or moretranscription factors selected from HNF1A, TCF-1, SIX5, HNF4A, SIN3A,ID1, and HNF1A are introduced into human pluripotent stem cells.

In particular, the FOXA1 gene and HNF1A (base sequence: SEQ ID NO: 11,amino acid sequence: SEQ ID NO: 12) are preferably introduced intopluripotent stem cells.

(Transcription Factor Required for Induction of Differentiation intoHematopoietic Cells)

A method of inducing differentiation into hematopoietic cells is asdescribed below.

Only one transcription factor, or two, three, four, five, six, or seventranscription factors selected from CDYL2, ETS2, SPI1, OVOL2, CDX2,CEBPB, and SALL4 are introduced into human pluripotent stem cells.

In particular, SPI1 (base sequence: SEQ ID NO: 18, amino acid sequence:SEQ ID NO: 19) is preferably introduced into pluripotent stem cells.

(Method of Introducing Target Gene into Genome of Pluripotent Stem Cell)

In a step of the method of the present invention, a method known per semay be used without any particular limitation as a method of introducingthe gene for the compound having an action of substantially removing orreducing an expression amount of a POU5F1 protein (or the gene for thecompound having an action of substantially removing or reducing H3K27me3modification) into the genome of the pluripotent stem cell. There may bepreferably used an expression cassette inserted between PiggyBactransposase recognition sequences (PB sequences) developed by Woltjen etal. (reference: Nature 458: 766-770, 2009.), which is a mechanism bywhich a gene to be introduced is actively incorporated into pluripotentstem cells (in particular, the genome of human ES cells). The expressioncassette is a system capable of efficiently establishing a geneticallymodified pluripotent stem cell line by introducing a drug selectioncassette (see WO 2017/131238 A1).

(Method of introducing Target Protein into Pluripotent Stem Cell)

In a step of the method of the present invention, a method known per semay be used as a method of introducing (transfecting) the compoundhaving an action of substantially removing or reducing an expressionamount of a POU5F1 protein (or the compound having an action ofsubstantially removing or reducing H3K27me3 modification) (inparticular, a protein) into the pluripotent stem cell, and examplesthereof may include: a method involving using a protein transfectionreagent; a method involving using a fusion protein having added theretoa cell-penetrating peptide; and a microinjection method.

The “cell-penetrating peptide” in the present invention is a peptidehaving a property of migrating into a cell, more specifically a propertyof penetrating a cell membrane, still more specifically a property ofpenetrating a cell membrane or a nuclear membrane to penetrate intocytoplasm or a nucleus. The amino acid sequence of the peptide is notparticularly limited, but examples thereof may include TAT(GRKKRRQRRRPQ: SEQ ID NO: 13), r8 {rrrrrrrr (D-form-R): SEQ ID NO: 14},and MPG-8 (βAFLGWLGAWGTMGWSPKKKRK: SEQ ID NO: 15).

The target protein encompasses the compound having an action ofsubstantially removing or reducing an expression amount of a POU5F1protein (or the compound having an action of substantially removing orreducing H3K27me3 modification) (in particular, a protein).

(Gene Knockout Method)

A gene knockout method is available as a method other than theforegoing. A “pluripotent stem cell in which a POU5F1 gene has beendisrupted” may be generated by the gene knockout method. The“pluripotent stem cell in which a POU5F1 gene has been disrupted” meansthat normal expression of the POU5F1 gene is inhibited due to artificialmodification of the sequence of a POU5F1 gene region, and as a result,the expression of POU5F1 is suppressed and a POU5F1 protein is notnormally expressed.

In addition, the “whole” in “modification or deletion of part or thewhole of the POU5F1 gene” refers to the protein-coding region of POU5F1genomic DNA.

In addition, the “part” refers to a region that is part of theprotein-coding region and that has a length required for inhibitingnormal expression of the POU5F1 gene.

Further, the “modification” refers to modification of the base sequenceof a target region in genomic DNA into another base sequence bysubstituting, deleting, inserting, and/or adding a single nucleotide ora plurality of nucleotides.

(Differentiation Induction Kit for Inducing Differentiation ofPluripotent Stem Cell into Desired Cell Type with High Efficiency)

A differentiation induction kit for efficiently inducing differentiationof a pluripotent stem cell into a desired cell type of the presentinvention (hereinafter sometimes referred to as “kit of the presentinvention”) includes any one or more of the following items (1) to (5)in addition to a transcription factor required for induction ofdifferentiation into the desired cell type and a Sendai virus vector.

(1) Pluripotent Stem Cell in which Expression Amount of POU5F1 Proteinhas been substantially removed or reduced and/or H3K27me3 Modificationhas been substantially removed or reduced

A user can easily induce differentiation into the desired cell type by,as described above, introducing a Sendai virus vector containing atranscription factor required for induction of differentiation into thedesired cell type into a pluripotent stem cell in which an expressionamount of a POU5F1 protein has been substantially removed or reducedand/or H3K27me3 modification has been substantially removed or reduced.

In addition, such pluripotent stem cell encompasses a pluripotent stemcell having a gene construct inducible with doxycycline or the likeinserted into the genome thereof so that a gene for a compound having anaction of substantially removing or reducing an expression amount of aPOU5F1 protein, a demethylase, or the like can be transiently forciblyexpressed therein.

(2) Gene for Compound Having Action of Substantially Removing orReducing Expression Amount of POU5F1 Protein and/or Demethylase Gene forKit of the Present Invention

The user can easily generate the pluripotent stem cell in which anexpression amount of a POU5F1 protein has been substantially removed orreduced and/or H3K27me3 modification has been substantially removed orreduced by adding a gene for a compound having an action ofsubstantially removing or reducing an expression amount of a POU5F1protein and/or a demethylase gene for a kit to a pluripotent stem cell.

Examples of anti-POU5F1 antibody gene may include, but not particularlylimited to, commercially available antibody genes.

Examples of the demethylase gene for a kit may include, but notparticularly limited to, mRNAs, DNAs, and proteins of demethylase genes(e.g., JMJD3c).

(3) Gene for Compound Having Action of Substantially Removing orReducing Expression Amount of POU5F1 Protein and/or Demethylase Gene,and Sendai Virus Vector Containing Transcription Factor Required forInduction of Differentiation into Desired Cell Type for Kit of thePresent Invention

The user can easily generate the pluripotent stem cell in which anexpression amount of a POU5F1 protein has been substantially removed orreduced and/or H3K27me3 modification has been substantially removed orreduced, and further, can induce differentiation thereof into thedesired cell type with high efficiency by adding a gene for a compoundhaving an action of substantially removing or reducing an expressionamount of a POU5F1 protein and/or a demethylase gene, and a Sendai virusvector containing a transcription factor required for induction ofdifferentiation into the desired cell type for a kit to a pluripotentstem cell.

Those genes may exist on one gene, or on separate genes. When the genesare present on separate genes, the gene for the compound having anaction of substantially removing or reducing an expression amount of aPOU5F1 protein (or the demethylase gene) and the Sendai virus vectorcontaining the transcription factor required for induction ofdifferentiation into the desired cell type may be added to thepluripotent stem cell simultaneously or at separate times.

(4) Compound Having Action of Substantially Removing or ReducingExpression Amount of POU5F1 Protein and/or Demethylase for Kit of thePresent Invention

The user can easily generate the pluripotent stem cell in which anexpression amount of a POU5F1 protein has been substantially removed orreduced and/or H3K27me3 modification has been substantially removed orreduced by adding a compound having an action of substantially removingor reducing an expression amount of a POU5F1 protein and/or ademethylase for a kit to the pluripotent stem cell.

(5) Gene Construct Carrying Gene for Compound Having Action ofSubstantially Removing or Reducing Expression Amount of POU5F1 Proteinand/or Demethylase Gene

The user can easily generate the pluripotent stem cell in which anexpression amount of a POU5F1 protein has been substantially removed orreduced and/or H3K27me3 modification has been substantially removed orreduced by introducing a gene construct carrying a gene for a compoundhaving an action of substantially removing or reducing an expressionamount of a POU5F1 protein and/or a demethylase gene into the genome ofa pluripotent stem cell.

The gene construct may contain a promoter sequence, a geneexpression-enhancing sequence, a marker gene, a reporter sequence, adrug resistance gene, and the like as required in addition to the genefor the compound having an action of substantially removing or reducingan expression amount of a POU5F1 protein and/or the demethylase gene.

A method of the present invention may be exemplified by, but notparticularly limited to, a method including any one of the followingsteps (1) to (8):

(1) a step of adding a gene for a compound having an action ofsubstantially removing or reducing an expression amount of a POU5F1protein and a Sendai virus vector containing a transcription factorrequired for induction of differentiation into the desired cell type toa pluripotent stem cell;

(2) a step of inserting a gene construct carrying a gene for a compoundhaving an action of substantially removing or reducing an expressionamount of a POU5F1 protein and a Sendai virus vector containing atranscription factor required for induction of differentiation into thedesired cell type into the genome of a pluripotent stem cell;

(3) a step of adding a Sendai virus vector containing a transcriptionfactor required for induction of differentiation into the desired celltype to a pluripotent stem cell in which an expression amount of aPOU5F1 protein has been substantially removed or reduced;

(4) a step of adding a Sendai virus vector containing a transcriptionfactor required for induction of differentiation into the desired celltype to a pluripotent stem cell in which a compound having an action ofsubstantially removing or reducing an expression amount of a POU5F1protein is forcibly expressed;

(5) a step of adding a compound having an action of substantiallyremoving or reducing an expression amount of a POU5F1 protein and aSendai virus vector containing a transcription factor required forinduction of differentiation into the desired cell type to a pluripotentstem cell;

(6) a step of adding a Sendai virus vector containing a transcriptionfactor required for induction of differentiation into the desired celltype to a pluripotent stem cell in which a POU5F1 gene has beendisrupted;

(7) a step of adding a gene for a compound having an action ofsubstantially removing or reducing an expression amount of a POU5F1protein, a demethylase gene, and a Sendai virus vector containing atranscription factor required for induction of differentiation into thedesired cell type to a pluripotent stem cell; and

(8) a step of adding a Sendai virus vector containing a transcriptionfactor required for induction of differentiation into the desired celltype to a pluripotent stem cell in which an expression amount of aPOU5F1 protein has been substantially removed or reduced and which has ahistone in which H3K27me3 modification has been substantially removed orreduced.

In the method of the present invention, any one of the followingpluripotent stem cells for differentiation into the desired cell typemay also be used:

(1) a pluripotent stem cell for differentiation into the desired celltype, in which an expression amount of a POU5F1 protein has beensubstantially removed or reduced;

(2) a pluripotent stem cell for differentiation into the desired celltype, in which a gene for a compound having an action of substantiallyremoving or reducing an expression amount of a POU5F1 protein isforcibly expressed;

(3) a pluripotent stem cell for differentiation into the desired celltype, which has a gene construct carrying a gene for a compound havingan action of substantially removing or reducing an expression amount ofa POU5F1 protein inserted into the genome thereof;

(4) a pluripotent stem cell for differentiation into the desired celltype, in which a POU5F1 gene has been disrupted; and

(5) a pluripotent stem cell for differentiation into the desired celltype, in which an expression amount of a POU5F1 protein has beensubstantially removed or reduced and which has a histone in whichH3K27me3 modification has been substantially removed or reduced.

EXAMPLES

The present invention is described below by way of Examples, but thepresent invention is by no means limited to Examples.

Example 1

(Differentiation into Skeletal Muscles)

In this Example, it was confirmed that, through the use of atemperature-sensitive Sendai virus vector expressing a human MYOD1 gene,hPSCs were able to be differentiated into skeletal muscles within 1week. In this Example, it was also confirmed that the addition ofsiPOU5F1, which suppressed the expression of a pluripotency gene POU5F1(also known as OCT4 or OCT3/4), further enhanced differentiationefficiency.

(Materials and Methods)

<Cell Culture>

An iPSC line derived from human adipose stem cells (iPSCs-ADSC) wasobtained from System Biosciences (Palo Alto). Cells were maintained asundifferentiated pluripotent cells in accordance with a standard hPSCculture method involving using StemFit basic02 (Ajinomoto) supplementedwith 100 ng/ml FGF2. The cells were cultured on a cell culture dishcoated with lamin-511 (iMatrix-511, Nippi). For skeletal muscledifferentiation, α-MEM (Thermo-Fisher) supplemented with 5% KSR(Thermo-Fisher) was used as a culture medium.

<Sendai Virus Vector>

A temperature-sensitive Sendai virus vector expressing human MYOD1(SeV18+hMYOD1/TS15ΔF) was custom-made by ID Pharma on contract. ThisSendai virus vector is F protein-deficient, and hence isnontransmissible (Inoue et al., J Virol. 77.5: 3238-3246, 2003). ThisSendai virus vector is temperature-sensitive, and this virus functionsat 33° C. and is inactivated at 37° C. (Ban et al., Proc Natl Acad SciUSA. 2011; 108(34): 14234-14239). A stock solution of the Sendai virusvector was diluted to 1×10⁴ cell-infecting units (CIU)/μl.

<Phosphospermine-Bound siPOU5F1>

In order to suppress the expression of human POU5F1 in the hPSCs, smallinterfering RNAs (siRNAs) against human POU5F1 were designed and used.In order to introduce the siRNAs without using lipofection or any othercationic lipid-based transfection reagent, the siRNAs were bound withphosphospermine (Paris et al., Molecular Pharmaceutics 2012. 9:3464-3475). Sequences used are a human POU5F1 sense strand:5′-GCCCGAAAGAGAAAGCGAAdT*dT-3′ (SEQ ID NO: 16) and a human POU5F1antisense strand: 5′-UUCGCUUUCUCUUUCGGGCdCdT-3′ (SEQ ID NO: 17). Thesense strand has 19 RNA bases and 2 DNA bases having added thereto 30spermine molecules at the site indicated by *. The antisense strand has19 RNA bases and 2 DNA bases. The sense strand and the antisense strandwere annealed, and stored as a 100 μM stock solution.

<Procedure>

(1) On Day 1, the human iPSCs (iPSCs-ADSC line) were plated in a 24-wellplate or a 4-well plate at 1.0×10⁵ cells in 250 μl of the medium perwell. The culture medium used was α-MEM (Thermo-Fisher) supplementedwith 5% KSR (Thermo-Fisher). Immediately after the plating, the Sendaivirus vector encoding the human MYOD1 gene was added to the cell culturemedium at a multiplicity of infection (MOI) of 2.5. In this Example, 25μl of a Sendai virus solution containing 2.5×10⁵ CIU was added to 250 μlof the medium containing 1.0×10⁵ cells. One hour after the addition ofthe Sendai virus vector, 1 ml of a culture solution was added to a totalamount of 1.275 ml per well. The cells were cultured in a CO₂ incubatorat 33° C., a permissive temperature for viral replication and geneexpression. The culture medium (1 ml/well) was changed daily.

(2) On Day 2, 4 μl of phosphospermine-siPOU5F1 (100 μM stock solution)was added to 1 ml of the medium per well at a final concentration of 400nM.

(3) On Day 4, the temperature of the CO₂ incubator was changed to 37°C., a non-permissive temperature for viral replication and geneexpression.

(4) On Day 5, spindle-shaped cells serving as a clear indication ofskeletal muscle differentiation were observed.

(5) On Day 6, the cells were fixed, and the efficiency of celldifferentiation was evaluated using immunostaining. The immunostainingwas performed by incubating the fixed cells overnight using ananti-myosin heavy chain (MHC) antibody (R&D systems) in 1:400 dilution.The cells were incubated for 1 hour using Alexa fluor 555 goatanti-mouse IgG as a secondary antibody in 1:200 dilution.

In FIG. 1A, a typical experimental procedure of the method ofdifferentiating hPSCs into skeletal muscles according to the presentinvention is illustrated.

(Results)

FIG. 1B is an example of a microscopic image (10× objective lens) of thecells immunostained with anti-myosin heavy chain (MHC) (red signal),which is specific for mature skeletal muscles. The cells were stainedwith DAPI (green signal) for visualizing the nuclei of all cells. Anenlarged microscopic image (20× objective lens) of the cells is shown inFIG. 10. Highly efficient formation of skeletal muscle cells was shownin visual inspection of the immunostaining result. By countingDAPI-positive cells and MHC-positive cells in a total of five images, itwas found the average fraction (average differentiation efficiency) ofthe MHC-positive cells in the DAPI-positive cells was 84.7% (528cells/623 cells). This average differentiation efficiency was thehighest efficiency of skeletal muscle differentiation from hPSCs in thehitherto reported results. In addition, in the method of the presentinvention, an efficiency of skeletal muscle differentiation of 90% ormore was observed.

As apparent from the above-mentioned results, the method of the presentinvention was able to achieve differentiation of hPSCs into skeletalmuscle cells rapidly (5 days), efficiently and homogeneously (up toabout 85% of MHC-positive skeletal muscle cells in all cells duringculture), and simply (only one time of treatment with the Sendai virusvector, and only one time of treatment with phosphospermine-siPOU5F1).In Table 1, a comparison between the method of the present invention andrelated-art methods is shown.

TABLE 1 Efficiency (% Speed MHC-positive Method (days) cells) Effect Thepresent invention 5 ~85% One time of Sendai virus infection; one time ofphosphospermine-siPOU5F1 treatment. One kind of cell culture medium.Synthetic mRNA cocktail 5 ~65% Five times of transfection (Akiyama etal., with synthetic mRNA cocktail. Development. 2016; 143: Two kinds ofcell culture 3674-3685) media. Successive changes in cell 20 ~70% Threekinds of cell culture culture environment media. (medium). AMSBIO(amsbio.com) Skeletal muscle differentiation kit

As apparent from the foregoing, as compared to the related-art methods,the method of the present invention is a method capable of achieving askeletal muscle cell differentiation efficiency of from about 71% ormore to about 90% or less, from about 73% or more to about 90% or less,from about 75% or more to about 90% or less, from about 78% or more toabout 90% or less, from about 80% or more to about 90% or less, fromabout 82% or more to about 90% or less, from about 83% or more to about90% or less, from about 84% or more to about 90% or less, from about 85%to about 90% or less, about 85%, about 90%, or about 90% or more withone kind of medium and through one time of transcription factorintroduction. Further, the method of the present invention is a methodcapable of achieving the above-mentioned differentiation efficiencywithin 12 days, within 10 days, within 9 days, within 8 days, within 7days, within 6 days, or within 5 days.

Example 2

(Differentiation into Motor Neurons)

In this Example, it was confirmed that, through the use of atemperature-sensitive Sendai virus vector expressing a human NGN3 gene,hPSCs were able to be differentiated into cholinergic and/or motorneurons within 1 week.

(Materials and Methods)

<Cell Culture>

An iPSC line derived from human adipose stem cells (iPSCs-ADSC) wasobtained from System Biosciences (Palo Alto). Cells were maintained asundifferentiated pluripotent cells in accordance with a standard hPSCculture method involving using StemFit basic02 (Ajinomoto) supplementedwith 100 ng/ml FGF2. The cells were cultured on a cell culture dishcoated with lamin-511 (iMatrix-511, Nippi). For motor neurondifferentiation, a 1:1 mixture of DMEM/F12 HAM and Neurobasal medium wasused as a cell culture medium. The medium was supplemented with1×N2/B27, dorsomorphin (final concentration: 3.3 μM), SB431542 (finalconcentration: 3.3 μM), and forskolin (final concentration: 3.3 μM).

<Sendai Virus Vector>

A temperature-sensitive Sendai virus vector expressing human NGN3(SeV18+hNGN3/TS15ΔF) was custom-made by ID Pharma on contract. ThisSendai virus vector is F protein-deficient, and hence isnontransmissible. This Sendai virus vector is temperature-sensitive, andthis virus functions at 33° C. and is inactivated at 37° C. A stocksolution of the Sendai virus vector was diluted to 1×10⁴ CIU/μl.

<Procedure>

(1) On Day 1, the human iPSCs (iPSCs-ADSC line) were plated in a 24-wellplate or a 4-well plate at 1.0×10⁵ cells in 250 μl of the medium perwell. The culture medium is as described above (1:1 mixture of DMEM/F12HAM and Neurobasal medium supplemented with 1×N2/B27, dorsomorphin,SB431542, and forskolin). Immediately after the plating, the Sendaivirus vector encoding the human NGN3 gene was added to the cell culturemedium at a multiplicity of infection (MOI) of 2.5. In this Example, 251μl of a Sendai virus solution containing 2.5×10⁵ CIU was added to 250 μlof the medium containing 1.0×10⁵ cells. One hour after the addition ofthe Sendai virus vector, 1 ml of a culture solution was added to a totalamount of 1.275 ml per well. The cells were cultured in a CO₂ incubatorat 33° C., a permissive temperature for viral replication and geneexpression. The culture medium (1 ml/well) was changed daily.

(2) On Day 3, the temperature of the CO₂ incubator was changed to 37°C., a non-permissive temperature for viral replication and geneexpression.

(3) As an optional procedure, on Day 4, the cells were passaged, andcultured on an ornithine/laminin-coated glass coverslip so thatdifferentiated neurons were easily visible under a microscope.

(4) On Day 6, the cells were fixed, and the efficiency of celldifferentiation was evaluated using immunostaining. The immunostainingwas performed by incubating the fixed cells overnight using ananti-tubulin β(TUBB3) antibody or anti-choline acetyltransferase (ChAT)antibody.

In FIG. 2A, a typical experimental procedure of the method ofdifferentiating hPSCs into neurons according to the present invention isillustrated.

(Results)

FIG. 2B to FIG. 2D are examples of microscopic images (20× objectivelens) of the immunostained cells. FIG. 2B is an image of the cellsstained with DAPI (blue signal) for visualizing the nuclei of all cells.FIG. 2C is an image of the cells immunostained with anti-β3-tubulin(TUBB3) antibody (red signal), which is specific for mature neurons.FIG. 2D is an image of the cells immunostained with anti-cholineacetyltransferase (ChAT) antibody (green signal), which is specific formotor neurons. FIG. 2E is a synthetic image of FIG. 2C and FIG. 2D. Bycounting DAPI-positive cells and TUBB3-positive cells in a total of fiveimages, it was found that the average fraction of the TUBB3-positivecells in the DAPI-positive cells (average differentiation efficiency)was 89.5% (205 cells/229 cells). This result of average differentiationefficiency shows that the method of the present invention can produce upto about 90% of neurons from hPSCs. By counting ChAT-positive cells andTUBB3-positive cells in a total of five images, it was found that theaverage fraction of the ChAT-positive cells in the TUBB3-positive cellswas 93.2% (191 cells/205 cells). This result shows that most of theneurons produced using the combination of the Sendai virus vectorexpressing NGN3 and the differentiation medium are motor neurons. Theefficiency was confirmed to be the highest efficiency of motor neurondifferentiation from hPSCs in the hitherto reported results.

As apparent from the above-mentioned results, the method of the presentinvention was able to achieve differentiation of hPSCs into motorneurons rapidly (5 days), efficiently and homogeneously (up to about 90%of TUBB3-positive neurons in all cells during culture), and simply (onlyone time of treatment with the Sendai virus vector). In Table 2, acomparison between the method of the present invention and related-artmethods is shown.

TABLE 2 Efficiency (% TUBB3-positive Method Speed (days) cells) EffectThe present invention 5 ~90% One time of Sendai virus infection. Onekind of cell culture medium. Synthetic mRNA cocktail 5 ~90% Two to fourtimes of (Goparaju et al., transfection with Scientific Reports.synthetic mRNA cocktail. 2017; 7: 42367) Two kinds of cell culturemedia. Successive changes in 10 ~75% Five kinds of cell culture cellculture media. environment (medium) (Chambers et al. 2012. NatureBiotech 7: 715.)

As apparent from the foregoing, as compared to the related-art methods,the method of the present invention is a method capable of achieving amotor neuron differentiation efficiency of from about 76% or more toabout 90% or less, from about 78% or more to about 90% or less, fromabout 80% or more to about 90% or less, from about 82% or more to about90% or less, from about 84% or more to about 90% or less, from about 86%or more to about 90% or less, from about 88% or more to about 90% orless, or about 90% with one kind of medium and through one time oftranscription factor introduction. Further, the method of the presentinvention is a method capable of achieving the above-mentioneddifferentiation efficiency within 12 days, within 10 days, within 9days, within 8 days, within 7 days, within 6 days, or within 5 days.

Example 3

(Differentiation into Liver Cells)

In this Example, it was confirmed that a temperature-sensitive Sendaivirus vector expressing a human FOXA1 gene and/or an HNF1A gene was ableto differentiate hPSCs into liver cells.

(Materials and Methods)

<Cell Culture>

An iPSC line derived from human adipose stem cells (iPSCs-ADSC) wasobtained from System Biosciences (Palo Alto). Cells were maintained asundifferentiated pluripotent cells in accordance with a standard hPSCculture method involving using StemFit basic02 (Ajinomoto) supplementedwith 100 ng/ml FGF2. The cells were cultured on a cell culture dishcoated with lamin-511 (iMatrix-511, Nippi). For liver celldifferentiation, media described in the literature (Hay et al., StemCells. 2008, 26: 894-902 and Kajiwara et al., Proc Natl Acad Sci USA.2012, 109: 14716) were used. Both cases in the literature took 18 daysfor differentiation, and used four kinds of cell culture media, i.e.,priming medium A (1 day), priming medium B (3 days), differentiationmedium (7 days), and maturation medium (7 days). On the other hand, asdescribed below, the method of the present invention took only 8 daysfor differentiation, and used only two kinds of differentiation media,i.e., differentiation medium (5 days) and maturation medium (1 day).

<Sendai Virus Vector>

A temperature-sensitive Sendai virus vector expressing human HNF1A(SeV18+hHNF1A/TS15ΔF) and a temperature-sensitive Sendai virus vectorexpressing FOXA1 (SeV18+hFOXA1/TS15ΔF) were custom-made by ID Pharma oncontract. This Sendai virus vector is F protein-deficient, and hence isnontransmissible. This Sendai virus vector is temperature-sensitive, andthis virus functions at 33° C. and is inactivated at 37° C. A stocksolution of the Sendai virus vector was diluted to 1×10⁴ CIU/μl.

<Phosphospermine-Bound siPOU5F1>

In order to suppress the expression of human POU5F1 in the hPSCs, smallinterfering RNAs (siRNAs) against human POU5F1 were designed and used.In order to introduce the siRNAs without using lipofection or any othercationic lipid-based transfection reagent, the siRNAs were bound withphosphospermine (Paris et al., Molecular Pharmaceutics 2012. 9:3464-3475). Sequences used are a human POU5F1 sense strand:5′-GCCCGAAAGAGAAAGCGAAdT*dT-3′ (SEQ ID NO: 16) and a human POU5F1antisense strand: 5′-UUCGCUUUCUCUUUCGGGCdCdT-3′ (SEQ ID NO: 17). Thesense strand has 19 RNA bases and 2 DNA bases having added thereto 30spermine molecules at the site indicated by *. The antisense strand has19 RNA bases and 2 DNA bases. The sense strand and the antisense strandwere annealed, and stored as a 100 μM stock solution.

<Procedure>

(1) On Day 1, the human iPSCs (iPSCs-ADSC line) were plated in a 24-wellplate or a 4-well plate at 1.0×10⁵ cells in 250 μl of the medium perwell. The cell culture medium used was a ROCK inhibitor(Y27632)-containing StemFit (registered trademark in Japan) Basic02(Ajinomoto). Immediately after the plating, a mixture of the Sendaivirus vector encoding the human HNF1A gene (25 μl of a Sendai virussolution containing 2.5×10⁵ CIU) and the FOXA1 gene (25 μl of a Sendaivirus solution containing 2.5×10⁵ CIU) was added to the cell culturemedium. One hour after the addition of the Sendai virus vector, 1 ml ofa culture solution was added to a total amount of 1.30 ml per well. Thecells were cultured in a CO₂ incubator at 33° C., a permissivetemperature for viral replication and gene expression. The culturemedium (1 ml/well) was changed daily.

(2) On Day 2, 4 μl of phosphospermine-siPOU5F1 (100 μM stock solution)was added to 1 ml of the medium per well at a final concentration of 400nM.

(3) On Day 2, the cell culture medium was replaced with adifferentiation medium containing 1% DMSO.

(4) On Day 5, the temperature of the CO₂ incubator was changed to 37°C., a non-permissive temperature for viral replication and geneexpression.

(5) On Day 7, the cell culture medium was replaced with a maturationmedium containing L15 medium supplemented with 20 ng/ml hepatocytegrowth factor (HGF), 20 ng/ml oncostatin M (OSM), 1 μM dexamethasone, 10μM SB431542, 10 μM ROCK inhibitor, and 0.1 mg/ml ascorbic acid.

(6) On Day 8, the cells were fixed, and the efficiency of celldifferentiation was evaluated using immunostaining. The immunostainingwas performed by incubating the fixed cells using an anti-albuminantibody.

In FIG. 3A, a typical experimental procedure of the method ofdifferentiating hPSCs into liver cells according to the presentinvention is illustrated.

(Results)

It was confirmed that the method of the present invention was able toachieve differentiation of hPSCs into liver cells rapidly (8 days),efficiently and homogeneously, and simply (only one time of treatmentwith the Sendai virus vector, and only one time of treatment withphosphospermine-siPOU5F1) with the two kinds of media (see FIG. 3B toFIG. 3E).

Example 4

(Differentiation into Hematopoietic Cells)

In this Example, it is confirmed that a temperature-sensitive Sendaivirus vector expressing a human SPI1 gene can differentiate hPSCs intohematopoietic cells.

(Materials and Methods)

<Cell Culture>

An iPSC line derived from human adipose stem cells (iPSCs-ADSC) wasobtained from System Biosciences (Palo Alto). Cells were maintained asundifferentiated pluripotent cells in accordance with a standard hPSCculture method involving using StemFit basic02 (Ajinomoto) supplementedwith 100 ng/ml FGF2. The cells were cultured on a cell culture dishcoated with lamin-511 (iMatrix-511, Nippi). For hematopoietic celldifferentiation, α-MEM (Thermo-Fisher) supplemented with 5% KSR(Thermo-Fisher) was used as a culture medium.

<Sendai Virus Vector>

A temperature-sensitive Sendai virus vector expressing human SPI1(SeV18+hSPI1/TS15ΔF) was custom-made by ID Pharma on contract. ThisSendai virus vector is F protein-deficient, and hence isnontransmissible. This Sendai virus vector is temperature-sensitive, andthis virus functions at 33° C. and is inactivated at 37° C. A stocksolution of the Sendai virus vector was diluted to 1×10⁴ CIU/μl.

<Phosphospermine-Bound siPOU5F1>

In order to suppress the expression of human POU5F1 in the hPSCs, smallinterfering RNAs (siRNAs) against human POU5F1 were designed and used.In order to introduce the siRNAs without using lipofection or any othercationic lipid-based transfection reagent, the siRNAs were bound withphosphospermine (Paris et al., Molecular Pharmaceutics 2012. 9:3464-3475). Sequences used are a human POU5F1 sense strand:5′-GCCCGAAAGAGAAAGCGAAdT*dT-3′ (SEQ ID NO: 16) and a human POU5F1antisense strand: 5′-UUCGCUUUCUCUUUCGGGCdCdT-3′ (SEQ ID NO: 17). Thesense strand has 19 RNA bases and 2 DNA bases having added thereto 30spermine molecules at the site indicated by *. The antisense strand has19 RNA bases and 2 DNA bases. The sense strand and the antisense strandwere annealed, and stored as a 100 μM stock solution.

<Procedure>

(1) On Day 1, the human iPSCs (iPSCs-ADSC line) are plated in a 24-wellplate or a 4-well plate at 1.0×10⁵ cells in 250 μl of the medium perwell. The cell culture medium used is α-MEM (Thermo-Fisher) supplementedwith 5% KSR (Thermo-Fisher). Immediately after the plating, the Sendaivirus vector encoding the human SPI1 gene is added to the cell culturemedium at a multiplicity of infection (MOI) of 2.5. In this Example, 25μl of a Sendai virus solution containing 2.5×10⁵ CIU is added to 250 μlof the medium containing 1.0×10⁵ cells. One hour after the addition ofthe Sendai virus vector, 1 ml of a culture solution is added to a totalamount of 1.275 ml per well. The cells are cultured in a CO₂ incubatorat 33° C., a permissive temperature for viral replication and geneexpression. The culture medium (1 ml/well) is changed daily.

(2) On Day 2, 4 μl of phosphospermine-siPOU5F1 (100 μM stock solution)is added to 1 ml of the medium per well at a final concentration of 400nM.

(3) On Day 4, the temperature of the CO₂ incubator is changed to 37° C.,a non-permissive temperature for viral replication and gene expression.

(4) On Day 6, the cells are fixed, and the efficiency of celldifferentiation is evaluated using immunostaining. The immunostaining isperformed by incubating the fixed cells using an anti-CD45 antibody.

(Results)

It can be confirmed that the method of the present invention can achievedifferentiation of hPSCs into hematopoietic CD45-positive cells rapidly(5 days), efficiently and homogeneously, and simply (only one time oftreatment with the Sendai virus vector, and only one time of treatmentwith phosphospermine-siPOU5F1).

Example 5

(Differentiation into Dopaminergic Neurons)

In this Example, it is confirmed that a temperature-sensitive Sendaivirus vector expressing a human FOXA1 gene can differentiate hPSCs intodopaminergic neurons within 1 week.

(Materials and Methods)

<Cell Culture>

An iPSC line derived from human adipose stem cells (iPSCs-ADSC) wasobtained from System Biosciences (Palo Alto). Cells were maintained asundifferentiated pluripotent cells in accordance with a standard hPSCculture method involving using StemFit basic02 (Ajinomoto) supplementedwith 100 ng/ml FGF2. The cells were cultured on a cell culture dishcoated with lamin-511 (iMatrix-511, Nippi).

For dopaminergic neuron differentiation, a 1:1 mixture of DMEM/F12 HAMand Neurobasal medium was used as a cell culture medium. The medium wassupplemented with 1×N2/B27 (without vitamin A and retinoic acid), BDNF(20 ng/ml), GDNF (20 ng/ml), TGF-β3 (1 ng/ml), ascorbic acid (finalconcentration: 0.2 mM), and cAMP (final concentration: 0.5 mM).

<Sendai Virus Vector>

A temperature-sensitive Sendai virus vector expressing human FOXA1(SeV18+hFOXA1/TS15ΔF) was custom-made by ID Pharma on contract. ThisSendai virus vector is F protein-deficient, and hence isnontransmissible. This Sendai virus vector is temperature-sensitive, andthis virus functions at 33° C. and is inactivated at 37° C. A stocksolution of the Sendai virus vector was diluted to 1×10⁴ CIU/μl.

<Procedure>

(1) On Day 1, the human iPSCs (iPSCs-ADSC line) are plated in a 24-wellplate or a 4-well plate at 1.0×10⁵ cells in 250 μl of the medium perwell. The above-mentioned culture medium is used (1:1 mixture ofDMEM/F12 HAM and Neurobasal medium supplemented with 1×N2/27, BDNF,GDNF, TGF-β3, ascorbic acid, and cAMP). Immediately after the plating,the Sendai virus vector encoding the human FOXA1 gene is added to thecell culture medium at a multiplicity of infection (MOI) of 2.5. In thisExample, 25 μl of a Sendai virus solution containing 2.5×10⁵ CIU isadded to 250 μl of the medium containing 1.0×10⁵ cells. One hour afterthe addition of the Sendai virus vector, 1 ml of a culture solution isadded to a total amount of 1.275 ml per well. The cells are cultured ina CO₂ incubator at 33° C., a permissive temperature for viralreplication and gene expression. The culture medium (1 ml/well) ischanged daily.

(2) On Day 3, the temperature of the CO₂ incubator is changed to 37° C.,a non-permissive temperature for viral replication and gene expression.

(3) As an optional procedure, on Day 4, the cells are passaged, andcultured on an ornithine/laminin-coated glass coverslip so thatdifferentiated neurons are easily visible under a microscope.

(4) On Day 6, the cells are fixed, and the efficiency of celldifferentiation is evaluated using immunostaining. The immunostaining isperformed by incubating the fixed cells overnight using an anti-tubulinβ3 (TUBB3) antibody and tyrosine hydroxylase (TH).

(Results)

It is confirmed that the method of the present invention can achievedifferentiation of hPSCs into TH-positive dopaminergic neurons rapidly(5 days), efficiently and homogeneously, and simply (only one time oftreatment with the Sendai virus vector).

According to the present invention, the method of inducingdifferentiation of a pluripotent stem cell into a desired cell typewithin a short period of time and with high efficiency can be provided.

What is claimed is:
 1. A method of differentiating a pluripotent stemcell into a skeletal muscle cell, comprising: a) adding a Sendai virusvector encoding a transcription factor MYOD1 to a pluripotent stem cellin a cell culture medium; b) culturing the pluripotent stem cell todifferentiate the pluripotent stem cell into the skeletal muscle cell;and c) adding a nucleic acid encoding a compound having an action ofsubstantially removing or reducing an expression amount of POU5F1protein to the pluripotent stem cell, wherein differentiation efficiencyof the skeletal muscle cell is 75% or more.
 2. The method according toclaim 1, wherein the nucleic acid encoding the compound having an actionof substantially removing or reducing an expression amount of a POU5F1protein encodes siRNA against POU5F1.
 3. The method according to claim1, wherein the Sendai virus vector is temperature-sensitive.
 4. Themethod according to claim 1, wherein the adding the Sendai virus vectorencoding the transcription factor MYOD1 to the pluripotent stem cell inthe cell culture medium is performed once.
 5. The method according toclaim 1, wherein differentiation efficiency of the skeletal muscle cellis 75% or more, and wherein one kind of cell culture medium is used.